The objective of Project 1 is to characterize the important role of repressive histone methylation in nucleus accumbens (NAc) and prefrontal cortex (PFC) in mediating stimulant and opiate addiction. We have demonstrated downregulation of dimethylation of Lys 9 of histone H3 (H3K9me2) in NAc and PFC after stimulant or opiate exposure, effects mediated via the repression of G9a, a histone methyltransferase which catalyzes this epigenetic mark. We have also demonstrated that such adaptations, which occur with investigator- or self-administered drug, increase behavioral responses to both drugs of abuse. We will now extend our considerable preliminary findings in several ways. We will utilize ChlP-Seq to map H3K9me2 binding genome-wide in response to stimulant or opiate self-administration over a broad time course. While global levels of H3K9me2 are reduced in NAc by drug exposure, many individual genes show induction of G9a/H3K9me2 binding, indicating the existence of complex mechanisms that control chromatin modifications at particular genes. Our hypothesis is that the chromatin landscape at individual genes helps determine which genes show reduced vs. increased H3K9me2 binding overlaid on global decreases in the enzymatic machinery involved. In conjunction with the Chromatin and Gene Analysis Core, we will also gain crucial information concerning the relationship between H3K9me2 and several other chromatin modifications in the NAc and PFC in vivo, including the role of DNA methylation and of several histone deacetylases, and better understand why these various chromatin changes are long-lasting only at a subset of genes after a course of drug administration. Another major effort is to define the molecular mechanisms by which chronic stimulants or opiates repress G9a expression in NAc and PFC; we have highly novel evidence implicating the beta- catenin transcriptional network in this phenomenon. Finally, we will focus on a small number of target genes- which encode synaptic proteins-for H3K9me2 and related chromatin modifications and characterize their involvement in addiction-related phenomena. Working with Project 4, we will validate key findings from animal models in the NAc and PFC of addicted humans, emphasizing the translational nature of our PPG. RELEVANCE (See instmctions): Addiction remains one of the worid's greatest public health problems, yet its pathophysiology remains incompletely understood and available treatments for addictions to various drugs of abuse are inadequately effective for most people. We believe that the most effective way of eventually developing definitive treatments and cures for addiction rests in part in a better understanding of its underiying neurobiology.